The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis has led to a critical need for new therapeutic agents against tuberculosis. M. tuberculosis is endowed with a diverse range of glycosylphosphatidylinositides such as phosphatidylinositol mannosides (PIMs), lipomannan (LM) and lipoarabinomannan (LAM), which play an important role in the physiology as well as the immunopathogenesis of mycobacterial infection. Enormous progress has been made in delineating the structure and biosynthesis of PIMs, but until recently the enzymes and steps leading to LM/LAM biosynthesis were not known. The elucidation of the catalytic steps involved in the synthesis of these molecules will help define one of the most complex biosynthetic pathways of mycobacteria in addition to providing new targets for the identification of novel anti-mycobacterial agents. Mutants defective in their synthesis should also enable a precise contribution of these molecules to the immunopathogenesis of tuberculosis. We have recently identified two genes encoding mannosyltransferases responsible for the terminal steps of LM/LAM biosynthesis. In silico analysis of the genetic locale of these genes revealed a possible PIM/LM/LAM biosynthetic gene cluster in the genome of M. tuberculosis. In order to define the PIM/LM/LAM biosynthetic pathways of mycobacteria, we have begun the characterization of genes from this cluster. Thus, the specific aims of this proposal are: 1. Characterization of an acyltransferase by genetic and biochemical approaches;2. Functional characterization of a protein potentially involved in the transport of PIMs across the plasma membrane. PUBLIC HEALTH RELEVANCE: Identification and characterization of a gene cluster for glycosylphosphatidylinositides of mycobacteria. Dissection of the glycosylphosphatidylinositide (GPI) pathway in mycobacteria will lead to the discovery of a novel class of essential enzymes of M. tuberculosis that may represent new targets for therapeutic intervention. In addition, the mutants defective in some aspects of GPI synthesis will help define the precise role of these molecules in immunopathogenesis.